色氨酸转运系统改造对大肠杆菌产L-色氨酸的影响

Effect of engineering of L-tryptophan transport system on L-tryptophan production in Escherichia coli

近年来,转运系统改造已经成为氨基酸菌株菌种改良的重要手段。本研究以工业生产菌Escherichia coli MT-01/p Trp-01为出发菌株,首先利用Red重组技术,在菌株MT-01/p Trp-01基因组上敲除了色氨酸吸收基因mtr,发酵结果表明,敲除敲除突变菌的L-色氨酸产量达到35.87 g/L,与出发菌株E.coli MT-01/p Trp-01相比提高了32%;在此基础上,进一步考察了三种不同启动子(Pr,Ptac,Pser A)控制下L-色氨酸分泌基因ydd G的差异表达对菌体生长及菌株产L-色氨酸的影响。结果表明,当采用组成型启动子tac时,ydd G基因的过表达菌株L-色氨酸的产量为41.01 g/L,比mtr敲除菌株E.coli MT-11/p Trp-01的产量提高了14.3%,当采用温度诱导型启动子Pr调控ydd G基因表达时,L-色氨酸的产量与mtr敲除菌株E.coli MT-11/p Trp-01的产量相比提高了9.3%,L-色氨酸的产量达到了39.22 g/L;而采用基因ser A的天然启动子调控ydd G表达时,菌体的生长受到了明显抑制,L-色氨酸产量仅有27.1 g/L的色氨酸。综上,大肠杆菌基因mtr的敲除和基因ydd G的过表达均可以有效提高工程菌株生产色氨酸的能力。

In recent years, the strategy of transport system modification has been widely employed for the development of amino acid production strains.In the present study ,the industrial production strain Escherichla coli MT-01/pTrp-01 was chose as the start strain, the L- tryptophan uptake gene of mtr knockout mutant strain were built by the method of Red homologous recombination, the fermentation results of the mtr mutant showed that the production of tryptophan was 35.87 g/L, which was 32% higher than that of the origin strain.Furthermore ,the L-tryptophan excretion gene of yddG was overexpressed at different levels by fusing with three different promoter （Pr, Ptac and PserA ） , and the L-tryptophan yield and the cell growth of gene yddG overexpression mutants were studied.The fermentation results showed that the yddG overexpression mutant fused with the constitutive promoter of tac increased the production of L-tryptophan to 41.01 g/L,which was 14.3% higher than that of the gene mtr knockout strain, the yddg overexpression mutant driven by the temperature inducible promoter Pr produced 39.22 g/L L-tryptophan, which was 9.3% higher than that of the gene mtr knockout strain. However, the yddg overexpression mutant driven by the promoter serA only produced 27.1 g/L L-tryptophan, and the cell growth of strain got significantly restrained.To sum up, overexpression of gene yddG and knockout of gene mtr are beneficial to improve the ability of engineering strains to produce L- tryptophan.